Department of Biology, McMaster University , Life Sciences Building, 1280 Main St. West, Hamilton, Ontario, Canada L8S 4K1.
Environ Sci Technol. 2014 Oct 7;48(19):11462-70. doi: 10.1021/es502794h. Epub 2014 Sep 9.
Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 10(2) to 1.0 × 10(7) genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay's ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection.
肠出血性大肠杆菌 O157:H7 是引起全球许多胃肠道疾病和溶血性尿毒症综合征暴发的罪魁祸首。监测食品和水源中的这种病原体是一个重要的公共卫生问题。高度保守的遗传标记，其特征是特定菌株，可以直接鉴定目标病原体。在这项研究中，我们使用 TaqMan 探针定量 PCR（qPCR），基于位于 ybiX 基因中的保守特征插入/缺失（CSI），检查了基于一种新的检测策略用于检测 O157:H7 血清型的致病性大肠杆菌菌株。qPCR 检测从 1.0×10(2)到 1.0×10(7)基因组拷贝呈线性，当针对 15 种非 O157:H7 大肠杆菌进行测试时，特异性为 O157:H7。该检测方法在存在竞争的大肠杆菌 K-12 时也保持检测灵敏度，在 1000 倍和 800 倍目标 DNA 过量的情况下，异源非目标 DNA 分别被添加，这表明该检测方法能够在存在高水平背景 DNA 的情况下检测到 O157:H7。因此，本研究验证了将菌株特异性 CSIs 用作病原体检测的新型诊断标记物。
